A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity Tyler O. Kirby, 2,3 Angel Rivera, 1,2 Daniel Rein, 1 Minghui Wang, 1 Ilya Ulasov, 1 Martina Breidenbach, 1 Manjula Kataram, 1 Juan L. Contreras, 5 Carlos Krumdieck, 4 Masato Yamamoto, 2 Marianne G. Rots, 6 Hidde J. Haisma, 6 Ronald D. Alvarez, 3 Parameshwar J. Mahasreshti, 1,2,3 and David T. Curiel 1,2 1 Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, 2 The Gene Therapy Center, Departments of 3 Obstetrics and Gynecology, 4 Nutrition Sciences, and 5 Surgery, University of Alabama at Birmingham, Birmingham, Alabama; and 6 Department of Therapeutic Gene Modulation, Groningen University Institute for Drug Exploration, Groningen, the Netherlands ABSTRACT Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which in- volves ex vivo evaluation of conditionally replicative adeno- virus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver. Experimental Design: The Krumdieck thin-slice tissue culture system was used to obtain and culture slices of tumor xenografts of ovarian cancer cell lines, human primary ovar- ian tumors, and human liver. We determined the viability of slices in culture over a period of 36 to 48 hours by ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt)]) (MTS) as- say. In vitro Hey cells, slices of Hey xenografts, and human ovarian tumor or human liver slices were infected with 500vp/cell of either replication competent wild-type adenovirus (Ad5/3wt), conditionally replicative adenovi- rus (Ad5/3cox-2), or the replication deficient adenovirus (Ad5/3luc1). At 12-, 24-, and 36-hour intervals, the repli- cation of adenoviruses in these slices was determined by quantitative reverse transcription-PCR of adenoviral E4 copy number. Results: Primary tumor slices were able to maintain viability for up to 48 hours after infection with nonreplica- tive virus (Ad5luc1). Infection of Hey xenografts with Ad5/ 3cox-2 showed replication consistent with that seen in Hey cells infected in an in vitro setting. Primary tumor slices showed replication of both Ad5/3wt and Ad5/3cox over a 36-hour time period. Human liver slices showed replication of Ad5/3wt but a relative reduction in replication of Ad5/ 3cox-2 indicative of conditional replication “liver off” phe- notype, thus predicting lower toxicity. Conclusions: The thin-slice model system represents a stringent method of ex vivo evaluation of novel replicative adenoviral vectors and allows assessment of human liver replication relative to human tumor replication. This is the first study to incorporate this system for evaluation of ther- apeutic efficacy and replicative specificity of conditionally replicative adenoviruses. Also, the study is the first to pro- vide a valid means for preclinical assay of potential condi- tionally replicative adenovirus-based hepatotoxicities, thus providing a powerful tool to determine therapeutic index for clinical translation of conditionally replicative adenoviruses. INTRODUCTION Conditionally replicative adenoviruses have emerged as a novel and promising approach for a range of advanced neo- plasms (1– 6). In this regard, direct translation of this approach from the laboratory to human clinical trials has proceeded at an unprecedented pace (2, 7–9). Whereas these studies have high- lighted the overall safety of this approach, only limited efficacy has been noted when conditionally replicative adenoviruses have been used as single modality agents (10 –12). On this basis, it is clear that substantial design advancements must proceed to allow full realization of the promise of conditionally replicative adenovirus agents. Indeed, the recent development of advanced generation conditionally replicative adenoviruses, which em- body enhanced infectivity (13–17), has established a rational framework for additional developmental strategies based on addressing the defined biological limitations of conditionally replicative adenovirus function. The development of conditionally replicative adenovirus agents has exploited available murine SCID/xenograft systems to study efficacy parameters (18). In this regard, human serotype adenoviruses adapted as conditionally replicative adenoviruses undergo only limited replication in a murine host background. This fact has severely limited any understanding of toxicity related to the replicative phenotype of current conditionally replicative adenoviruses. Additionally, issues related to vector- host interaction may not be characterizable in these immunod- Received 6/16/04; revised 8/30/04; accepted 9/20/04. Grant support: The National Institute of Health Grants (CA 091078) and Grants T32 CA091078 and NIH/National Cancer Institute P50 CA83591. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: David T. Curiel, Division of Human Gene Therapy, The Gene Therapy Center, University of Alabama at Birming- ham, BMR2-Room 502, 901 19th Street South, Birmingham, AL 35294- 2172.Phone:(205) 934-8627;Fax:(205) 975-7949;E-mail:david.curiel@ ccc.uab.edu. ©2004 American Association for Cancer Research. 8697 Vol. 10, 8697– 8703, December 15, 2004 Clinical Cancer Research Downloaded from http://aacrjournals.org/clincancerres/article-pdf/10/24/8697/1923300/zdf02404008697.pdf by guest on 13 June 2022